Regular Paper
Roles of the Cyclic Electron Flow Around PSI (CEF-PSI) and
O2-Dependent Alternative Pathways in Regulation of the
Photosynthetic Electron Flow in Short-Term Fluctuating
Light in Arabidopsis thaliana
Masaru Kono*, Ko Noguchi and Ichiro Terashima
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
*Corresponding author: Email, [email protected]; Fax, +81-3-5841-4465.
(Received January 7, 2014; Accepted February 6, 2014)
To assess the roles of the cyclic electron flow around PSI
(CEF-PSI) and O2-dependent alternative pathways including
the water–water cycle in fluctuating light, we grew the wild
type and pgr5 mutant of Arabidopsis thaliana in constant
light, and measured Chl fluorescence and P700 parameters
in their leaves in the fluctuating light alternating between
240 (HL) and 30 mmol photons m2 s2 (LL) every 2 min. At
20% O2, the photochemical quantum yield of PSII decreased,
in particular in the pgr5 plants, soon after the start of the
fluctuating light treatment. PSI of the pgr5 plants was markedly photoinhibited by this treatment for 42 min. Slight PSI
photoinhibition was also observed in the wild type. We measured energy sharing between PSII and PSI and estimated the
PSI and PSII electron transport rates (ETRs). pgr5 showed
larger energy allocation to PSI. In contrast to the wild
type, the ratio of the PSI to the PSII ETR in pgr5 was
higher in LL but lower in HL at 20% O2 due to PSI acceptor-side limitation. At 2.7% or 0% O2, the CEF-PSI of
the pgr5 plants was enhanced, the acceptor-side limitation
of PSI electron flow was released and PSI photoinhibition
was not observed. The results suggest that the light fluctuation is a potent stress to PSI and that the CEF-PSI is essential to protect PSI from this stress.
Keywords: Alternative electron flow Fluctuating light PGR5 Photoinhibition of PSI.
Abbreviations: Amax, maximum level of P700 signal in the
dark; CEF, cyclic electron flow; ETR, electron transport rate;
FQR, ferredoxin-plastoquinone reductase; Fv/Fm, maximum
quantum yield of PSII; HL, high light; LED, light-emitting
diode; LEF, linear electron flow; LL, low light; NDH, NADH
dehydrogenase-like complex; NPQ, non-photochemical
quenching; PPFD, photosynthetic photon flux density;
PTOX, plastid terminal oxidase; qE, energy-dependent
quenching; qI, photoinhibitory quenching; qL, fraction of
open PSII reaction centers; qT, state transition quenching;
ROS, reactive oxygen species; SP, saturation pulse; WT,
wild-type; WWC, water–water cycle.
Introduction
Even in open habitats, plants experience dynamic fluctuations of
light because of clouds. Understorey plants experience more frequent, short-term light fluctuations due to leaves and stems of
other plants above them in addition to clouds. Plants have to
cope with these light fluctuations of various time ranges. In constant low light, plants can use most of the light energy in driving
photochemistry. In contrast, in constant high light, energy transfer from antenna Chls to the PSII reaction center is suppressed
and the excess energy is dissipated as heat. This process prevents
photoinhibition. When the light intensity fluctuates between low
and high levels very rapidly, however, it is not possible for chloroplasts to switch the heat dissipation system on and off synchronously with the light fluctuation because both induction and
deactivation of the heat dissipation system require at least several minutes (Müller et al. 2001). Plants must have more rapid
systems to cope with very rapid light fluctuations.
Photosynthetic electron transport primarily occurs via a
linear pathway, in which electrons flow from water via PSII
and the Cyt b6/f complex to PSI and reduce NADP+ to
NADPH. The linear electron flow (LEF) generates the transmembrane electrochemical potential difference of H+, through
water splitting by PSII in the thylakoid lumen and translocation
of H+ across the thylakoid membrane by the Q cycle. The electrochemical potential difference thus produced drives the H+ATP synthase to produce ATP. Low pH in the thylakoid lumen
causes de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin and protonation of the PsbS protein, both of which
contribute to the heat dissipation, which can be measured
fluorometrically as the non-photochemical quenching (NPQ).
In addition to the LEF system, there are two PSI cyclic electron flow (CEF) systems (Shikanai 2007): the NADH dehydrogenase-like complex-dependent pathway (NDH-CEF; Burrows
et al. 1998, Shikanai et al. 1998, Peng et al. 2011, Yamamoto et al.
2011) and the ferredoxin-plastoquinone reductase pathway
(FQR-CEF; Munekage et al. 2002, Munekage et al. 2004,
DalCorso et al. 2008, Hertle et al. 2013). FQR-CEF involves the
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033, available online at www.pcp.oxfordjournals.org
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Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
Responses of alternative electron flows to fluctuating light
Cyt b6/f complex, plastocyanin, PSI, ferredoxin (Fd) and FQR.
PGR5 was identified as an essential component of the FQR-CEF
(Munekage et al. 2002; see below). Very recently, PGRL1 has
been proposed to be the elusive FQR (Hertle et al. 2013). In C4
plants, the CEFs around PSI (CEF-PSI), particularly the NDHCEF, operate to supply ATP to the CO2-concentrating mechanism (Takabayashi et al. 2005) as well as the Calvin–Benson
cycle. It is noteworthy that the NDH-CEF also involves ferredoxin (Okegawa et al. 2008, Johnson 2011, Yamamoto et al.
2011). It is often claimed that ATP and NADPH production
by LEF cannot meet the required ATP/NADPH ratio for the
photosynthetic carbon fixation by the Calvin–Benson cycle.
In particular, when photorespiration occurs at high rates, the
required ratio shifts from 3ATP/2NADPH towards 3.5ATP/
2NADPH, and thereby shortage of ATP may be more serious
(Allen 2002, Shikanai 2007). The CEF-PSI would contribute to
producing additional ATP. Another function of CEF-PSI is enhancement of the NPQ, through generating the electrochemical potential difference of H+ across the thylakoid membrane
(Munekage et al 2002).
Pseudo-cyclic electron flow, also called the water–water
cycle (WWC) (Asada 1999) or the Mehler–ascorbate peroxidase
(MAP) pathway (Schreiber et al. 1995), is the electron flow from
water via PSII, Cyt b6/f and PSI to molecular oxygen. Since the
redox potentials of the electron acceptors on the acceptor side
of the PSI complex are sufficiently low to reduce O2, electron
flow to O2 occurs, particularly when NADP+ is not available.
This results in the formation of reactive oxygen species (ROS),
such as O2– and H2O2 (Asada 1999). Superoxide dismutase and
ascorbate peroxidase in the WWC scavenge O2– and H2O2.
NADPH is used to regenerate ascorbate from monodehydroascorbate or dehydroascorbate. Summing up these reactions,
electrons are transferred from water to H2O2 to form water.
Thus, the WWC acts as a large electron sink (Asada 2000). The
WWC also generates the electrochemical potential difference of
H+ across the thylakoid membrane, which enhances the nonradiative dissipation of excess light energy observed as the increase in NPQ. Therefore, the WWC is also considered to play
roles in dissipation of excess light energy (Osmond and Grace
1995, Osmond et al. 1997, Asada 1999, Asada 2000, Foyer and
Noctor 2000, Miyake 2010). The CEF-PSI and WWC are thought
to protect plants from damage that occurs due to the overreduction of the thylakoids under stress conditions (Miyake
2010).
An Arabidopsis thaliana mutant, pgr5 (proton gradient regulation), was reported to have impaired electron transfer in
FQR-CEF (Munekage et al. 2002). In a screen using the Chl
fluorescence imaging technique, this mutant showed a phenotype similar to that of npq mutants (Shikanai et al. 1999). NPQ
measurements with this mutant showed an almost complete
absence of energy-dependent quenching (qE) at high irradiance
under steady-state photosynthesis (Munekage et al. 2002).
Nandha et al. (2007), however, reported that the capacity
for cyclic electron transport in pgr5 was comparable with
that of the wild-type (WT). They also showed that the electron
transport system in pgr5 was greatly reduced under most
conditions.
A recent study has proposed an important role for PGR5 in
protection of PSI under fluctuating growth light in A. thaliana
(Suorsa et al. 2012). The pgr5 mutant showed no growth when
grown in drastically fluctuating light, alternating low light for
5 min and high light for 1 min (Tikkanen et al. 2010, Suorsa et al.
2012). In a more moderately fluctuating growth light, the PSI
complex in this mutant was found to be photodamaged. These
authors also reported that, under constant growth light, the
pgr5 mutant did not show any visible growth phenotype irrespective of the growth irradiance levels. From these findings,
they argued that the pgr5 could not maintain the redox balance
of the electron transfer reactions in fluctuating light. However,
how the redox imbalance occurs in pgr5 and how the WT
plants cope with the drastically fluctuating light are still unclear.
The aim of this study was to examine photosynthetic responses of WT and pgr5 plants, both grown in continuous moderate light in the light period, to a fluctuating light using
simultaneous Chl fluorescence and P700 measurements under
the precise control of gas concentrations. The fluctuating light
adopted was alternation of low light for 2 min and high light for
2 min. Even for high light, a moderate level of photosynthetically
active photon flux density (PPFD) was chosen. We examined
whether photoinhibition of PSI occurred in the mature leaves
of the pgr5 plants in short-term experiments for up to 42 min.
Next, we tried to elucidate which of the photosynthetic alternative electron flows was impaired in the pgr5 plants through
examining the effects of O2 concentrations at various PPFDs.
Results
Responses of PSII and PSI quantum yields to
fluctuating and continuous light
Chl fluorescence and absorption changes at 830 nm in the intact
leaf were measured simultaneously using a Dual-PAM-100
(Walt GmbH). Changes in the PSII quantum yield, Y(II), in
mature leaves of the WT and pgr5 plants were measured in
the light regime that alternated between high light (HL) at
240 mmol photons m2 s1 for 2 min and low light (LL) at
30 mmol photons m2 s1 for 2 min, for a total of 42 min
(Fig. 1). The leaf was kept in a small hand-made chamber,
and CO2 and O2 gas concentrations in the chamber were regulated with mass-flow controllers. Unless otherwise stated, the
CO2 and O2 concentrations were 390 p.p.m. and 20%, respectively. In the leaves of WT plants grown in the constant light at
90–100 mmol photons m2 s1 for 8 h d1, Y(II) at the end of
each LL period decreased with the cycle, but Y(II) at the end of
each HL period did not change after attaining the steady value
of around 0.45. In pgr5, Y(II) in the LL period decreased with the
cycle more markedly than in the WT. Y(II) in the HL period also
decreased after the fifth cycle. In the last cycle, Y(II) in the LL
period became 0.4, approaching that in the HL period which
was around 0.3.
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
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M. Kono et al.
A
pgr5
WT
B
Y(II)
Y(I)
A
: LL
: HL
D
Y(II)
C
B
: LL
Y(II)
: HL
Time (min)
To compare the photosynthetic responses in fluctuating
light and those in continuous light, we measured changes in
Y(II) and the PSI quantum yield, Y(I), in constant HL at
240 mmol photons m2 s1 or LL at 30 mmol photos m2 s1
for 42 min (Fig. 2). When the plants that had been kept in the
dark for >30 min were exposed to the constant HL, Y(I) of the
WT increased for about 5 min and attained a steady level, while
that in LL gradually decreased. Y(II) in HL once decreased, attained a steady level, while that in LL decreased and attained
the peak value at around 5 min and then slightly decreased.
In the pgr5 plants, Y(I) in LL showed a transient similar to
that of the WT. Y(I) in HL, however, once decreased, increased
to the peak at around 10 min and then decreased very slightly.
Changes in Y(II) in pgr5 were similar to those in the WT. In HL,
both Y(I) and Y(II) in pgr5 were considerably lower than those
in the WT.
Light responses of the steady-state PSI and PSII
parameters at various PPFDs
Light responses of PSI and PSII parameters obtained from Chl
fluorescence and P700 signals were further analyzed (Fig. 3). For
energy captured by PSI pigments, the quantum yield of the PSI
photochemistry, Y(I), the quantum yield of non-photochemical
energy dissipation due to the donor-side limitation, Y(ND), and
that of the energy dissipation due to the acceptor-side
992
pgr5
WT
Y(I)
Y(ND)
Y(NA)
Y(I)
Yield
Fig. 1 Response of the photochemical quantum yield of PSII [Y(II)] of
the WT (A) and pgr5 (B) plants to the fluctuating light. The plants
were grown in a constant moderate light (100 mmol photons m2 s1)
for 8 h d1. The light alternating between HL at 240 mmol photons
m2 s1 for 2 min (open bars) and LL at 30 mmol photons m2 s1 for
2 min (grey bars) was applied to the leaf after the dark treatment for
30 min. The leaf lamina was sandwiched in a chamber. The air in the
chamber contained 20% O2 and 390 p.p.m. CO2. Each data point represents the mean (n = 5–6).
Fig. 2 Changes in the quantum yield of PSI [Y(I)] and PSII [Y(II)].
(A and B), Y(I); (C and D), Y(II). Constant light at a PPFD of
240 mmol photons m2 s1 (open symbols, HL) or at a PPFD of
30 mmol photons m2 s1 (filled symbols, LL) was applied for 42 min
after the 30 min dark treatment. Measurements were made at 20% O2
and 390 p.p.m. CO2. Each data point represents the mean (n = 4).
Y(ND)
Y(NA)
Y(II)
Y(II)
Y(NO)
Y(NO)
Y(NPQ)
Y(NPQ)
Yield
Time (min)
Time (min)
PPFD (μmol m
2
s
1)
PPFD (μmol m
2
s
1)
Fig. 3 Changes in the photosynthetic quantum yields of PSI and PSII
with the PPFD of constant light in the WT (filled symbols) and pgr5
(open symbols). For energy captured by PSI pigments, the quantum
yield of the PSI photochemistry, Y(I) (circle), the quantum yield of
non-photochemical energy dissipation due to the donor-side limitation, Y(ND) (square), and that of the energy dissipation due to the
acceptor-side limitation, Y(NA) (triangle), are indicated. The fluorescence parameters, the effective PSII quantum yield, Y(II) (circle), the
quantum yield of regulated energy dissipation, Y(NPQ) (triangle), and
that of non-regulated energy dissipation, Y(NO) (square) are shown.
Measurements were made at 20% O2 and 390 p.p.m. CO2. The values
represent the mean ± SD (n = 4–6).
limitation, Y(NA), were measured. The fluorescence parameters
measured included the effective PSII quantum yield, Y(II), the
quantum yield of regulated energy dissipation, Y(NPQ), and
that of non-regulated energy dissipation, Y(NO).
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
Responses of alternative electron flows to fluctuating light
Y(I) and Y(II) in both the WT and pgr5 decreased with the
increase in PPFD. In the WT, Y(NA) was greater than Y(ND)
at PPFDs <250 mmol photons m2 s1, while, above this level,
Y(NA) decreased and Y(ND) increased. In the WT, with the
increase in PPFD, Y(NPQ) markedly increased, while Y(NO)
increased only slightly. pgr5 showed trends very different
from those of the WT. Y(NA) of pgr5 was similar to that of
the WT up to 100 mmol photons m2 s1, but it markedly
increased with further increases in PPFD, causing a drastic decrease in Y(I) in pgr5 at PPFDs >150 mmol photons m2 s1.
Y(ND) in pgr5 was nearly zero over the entire PPFD range. These
results indicate that, at high PPFDs, the electron flow through
PSI in pgr5 was limited by the acceptor-side reactions.
Furthermore, the increase in Y(NPQ) was much less than that
in the WT, while Y(NO) markedly increased.
Effects of fluctuating light on photoinhibition of
photosystems and photosynthetic electron
transport
The maximum level of the P700 signal (full oxidation of P700) in
the dark (Amax) and the maximum quantum yield of PSII (Fv/
Fm) were measured before and after the treatment with constant
HL (240 mmol photons m2 s1) or fluctuating light (alternating
between HL at 240 mmol photons m2 s1 for 2 min and LL at
30 mmol photons m2 s1 for 2 min), both for 42 min (Fig. 4).
After the light treatments, plants were kept in the dark for
30 min and Amax and Fv/Fm were measured. Amax and Fv/
Fm were unchanged after the constant HL treatment for 42 min
from the levels before the treatment (Fig. 4a, c), indicating that
ΔAmax
B
Y(NA)30
Do the pgr5 plants show CEF-PSI activity in
constant light?
To investigate whether the pgr5 plants showed CEF-PSI activity
in constant light, we estimated the ETR through PSI [ETR(I)]
and PSII [ETR(II)] simultaneously. The photochemical
quantum yield of PSI, Y(I), may be expressed as
Y(I) = Y(LI) + Y(WWC) + Y(CEFI), where Y(LI), Y(WWC) and
Y(CEFI) are the quantum yields of the LEF through PSI, the
WWC and the CEF-PSI, respectively. Similarly, the photochemical quantum yield of PSII, Y(II), may be written as
C
Fv/Fm
D
1-qL30
Relative value
A
photoinhibition of photosystems hardly occurred. Y(NA) and 1 –
qL were measured in LL at 30 mmol photons m2 s1 for 5 min
before and after the 42 min light treatment. The data obtained at
the end of 5 min of LL are denoted as Y(NA)30 and 1 – qL30,
respectively. After the HL treatment, both Y(NA)30 and 1 –
qL30 in pgr5 were significantly higher than before (Fig. 4b, d),
indicating some damage to the acceptor side of PSI.
The WT showed small decreases both in Amax and in Fv/Fm
after the treatment with the fluctuating light treatment
(Fig. 4e, g). In contrast, Amax in the pgr5 plants after the
fluctuating light treatment decreased by 38%, while Fv/Fm
decreased only slightly. Although there were only small decreases in Amax and Fv/Fm in the WT, Y(NA)30 and 1 – qL30
after the fluctuating light treatment increased by 42% and 135%
(Fig. 4f, h), respectively, indicating some damage to the acceptor side of PSI and the competence of PSI in oxidizing the
intersystem chain. In pgr5, Y(NA)30 and 1 – qL30 increased by
94% and 332% after the fluctuating light treatment.
F
G
H
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42
min
bef
ore
42m
in
Relative value
E
Fig. 4 Effects of constant high light (a–d) and fluctuating light (e–h) on changes in photosynthetic parameters in leaves of the WT (filled bars)
and pgr5 (open bars). Following the light treatments for 42 min and dark treatment for 30 min, the functions of the PSI and PSII reaction centers
were determined as Amax and Fv/Fm. Y(NA)30 and 1 – qL30 were measured at the end of the low light treatment at a PPFD of 30 mmol photons
m2 s1 for 2 min just after the light treatments for 42 min. Measurements were made at 20% O2 and 390 p.p.m. CO2. Error bars represent the SD
(n = 6–8).
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
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M. Kono et al.
Y(II) = Y(LII) + Y(WWC), where Y(LII) is the quantum yield of
the LEF through PSII.
To obtain directly comparable ETR(I) and ETR(II) values, we
measured leaf absorptance, and estimated the share of absorbed
light energy allocated to PSII (fPSII). Fig. 5A shows the relationships between the gross O2 evolution in the air containing 5%
CO2 and the absorbed PPFD at low PPFDs calculated with the
absorptance values, measured in four leaves each of the WT and
pgr5, respectively. When compared at the same absorbed PPFD,
the O2 evolution rates for pgr5 leaves was always lower than that
for the WT leaves, indicating that the quantum yield of O2 evolution on the absorbed quantum basis was lower in the pgr5
leaves. However, Fv/Fm values in the leaves did not differ between
the WT and pgr5. Fig. 5B shows relationships between the quantum yield of gross O2 evolution [Y(O2)] and that of PSII photochemistry [Y(II)] in the same leaves used for Fig. 5A. The slope of
the line should be proportional to the share of absorbed light
energy allocated to PSII (fPSII). In pgr5, the slope was lower by
17.3% than in the WT. The share of absorbed light energy allocated to PSII was 35 ± 3.4% in pgr5, while the share in the WT was
47 ± 4.1%. With these values, it would be possible to calculate the
absolute rates of ETR(I) and ETR(II).
s 1)
2
(µmol m
Rate of gross O2 evolution
A
Absorbed PPFD (µmol m
2
In the WT plants, the ETR(I)/ETR(II) ratio was close to 1 at
PPFDs below 100 mmol photons m2 s1, and subsequently increased with the increase in PPFD. In contrast, the
ETR(I)/ETR(II) ratio in pgr5 was high at low PPFDs and
decreased with the increase in PPFD, and eventually became
close to 1 (Fig. 6). In accordance with the widely accepted view,
the contribution of the CEF-PSI increased with the increase
in PPFD in the WT, while pgr5 showed a contrasting
trend: the contribution of CEF-PSI decreased with the increase
in PPFD.
The ETR(I)/ETR(II) ratio would not be correct if there
were large changes in the energy share between PSII and PSI
due to the state transition. To examine the contribution of the
state transition to the change in the ETR(I)/ETR(II) ratio, we
determined the components of non-photochemical Chl fluorescence quenching, qN, from the relaxation kinetics of qN in
the dark. Fig. 7 shows the extents of qE, state transition
quenching (qT) and photoinihibitory quenching (qI) after
20 min of constant light at PPFDs of 30, 240 and 470 mmol
photons m2 s1. Both WT and pgr5 leaves exhibited substantial qE, although qE in the pgr5 leaf was much less than that in
WT plants, as was reported previously (Munekage et al. 2002,
Munekage et al. 2008). qT components were small compared
with qE in both plants. As the measurements were carried out
with red actinic light, there was no effect of chloroplast
avoidance movement on the apparent state transition
(Cazzaniga et al. 2013). Therefore, it is unlikely that the state
transition affected the ETR(I)/ETR(II) ratio very much.
Table 1 shows the Chl contents on a leaf area basis in WT
and pgr5 leaves. The Chl content was higher in the WT than in
pgr5. There were some differences in absorptance, reflecting the
difference in the Chl content. The Chl a/b ratio in the pgr5
leaves was greater than that in WT leaves by 0.4.
s 1)
Y(O2)
ETR(I)/ETR(II)
B
Y(II)
Fig. 5 Estimation of the share of absorbed light energy to PSII. (A)
Light–response curve of the photosynthetic O2 evolution in the leaf
disks at low PPFDs. The rate of gross O2 evolution was plotted against
absorbed PPFD, Fitted hyperbolic functions through the origin are
shown. (B) The relationship between the quantum yield of O2 evolution, Y(O2), and the photochemical yield of PSII, Y(II). Filled circle, WT;
open circle, pgr5. Error bars represent the SD (n = 4–6). Regression
lines through the origin are shown.
994
PPFD (μmol m
2
s
1)
Fig. 6 Changes in the ETR(I)/ETR(II) ratio in WT (filled circle) and
pgr5 (open circle) leaves as a function of the PPFD of constant light.
Y(I) and Y(II) were measured as in Fig. 3. Measurements were made at
20% O2 and 390 p.p.m. CO2. The values represent the mean ± SD
(n = 4–6).
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
Responses of alternative electron flows to fluctuating light
Chlorophyll fluorescence yield
A
Time (s)
NPQ components
B
pgr5
WT
qE
qE
qT
qT
qI
qI
PPFD (µmol m
2
s
1)
PPFD (µmol m
2
s
1)
Fig. 7 Dissection of the non-photochemical quenching into energy-dependent quenching (qE), state transition quenching (qT) and photoinhibition quenching (qI). (A) A typical Chl fluorescence trace obtained with a WT leaf after 30 min dark adaptation. The trace shows the changes
in fluorescence yield during (white bar) and after turning off the actinic light (black bar). (B) Components of non-photochemical Chl fluorescence quenching in the WT and pgr5. The different components of non-photochemical fluorescence quenching were derived from
semi-logarithmic plots of the dark relaxation of Fv after the light treatment at three PPFDs of 30, 240 and 470 mmol photons m2 s1.
Energy-dependent quenching (qE; circle) was attributed to the fast phase, quenching by state transition (qT; square) to the medium phase
and photoinhibitory quenching (qI; triangle) to the slow phase of relaxation. The quenching components were calculated from the amplitude of
the respective phases considering the relationship (1 qN) = (1 qE) (1 qT)(1 qI). Measurements were made at 20% O2 and 390 p.p.m.
CO2. The values represent the mean ± SD (n = 3).
Table 1 Chl a + b, Chl a/b in thylakoids and leaf absorptance in the
WT and pgr5 leaves
Genotype
Chl a + b (mg m2)
Chl a/b
WT
228 ± 23
3.38 ± 0.007
0.837 ± 0.0521
pgr5
190 ± 10*
3.73 ± 0.009*
0.823 ± 0.0742*
Leaf absorptance
Plants were grown at 90–100 mmol m2 s1 in a short-day photoperiod (8 h of
light, 16 h of dark) for 55 d.
Means ± SD (n = 3–5) are shown.
Light absorptance was measured with an integrating sphere. The light from the
Björkman-type lamp passing through a 635 nm red filter was used.
*P < 0.005 (t-test, WT vs. pgr5).
Does the WWC in the pgr5 plants function in
constant light?
Next, we investigated whether the pgr5 plants were able to
drive the WWC. In the WWC, the electron acceptor from PSI is
O2 and the extent of the electron flow to the WWC depends on
the O2 concentration (Miyake and Yokota 2000). The CEF-PSI
activity has been shown to increase at a low O2 concentration,
indicating suppression of the WWC by low O2 (Arnon and Chain
1975, Arnon and Chain 1979, Scheller 1996, Makino et al. 2002). If
the pgr5 plants possessed no WWC capacity, the activity of the
CEF-PSI would not be enhanced even at low O2 concentrations.
We measured the light dependence of the ETR(I)/ETR(II) ratio on
the O2 concentration in the chamber, namely at 20, 2.7 and 0%
O2 (Fig. 8). In the pgr5 plants, the ETR(I)/ETR(II) ratios at 2.7%
and 0% O2 were higher than that at 20% O2 when the PPFD was
>100 mmol photons m2 s1. Both the WT and pgr5 exhibited
the highest ETR(I)/ETR(II) ratios at 2.7% O2 at any PPFD below
300 mmol photons m2 s1. Therefore, it is likely that the pgr5
plants possessed the capacity for the WWC.
Response of the ETR(I)/ETR(II) ratio to fluctuating
light
Changes in the ETR(I)/ETR(II) ratio in fluctuating light are shown
in Fig. 9. The same fluctuating light regime as used for the data in
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20%
2.7%
ETR(I)/ETR(II)
ETR(I)/ETR(II)
A
0%
20%
2.7%
ETR(I)/ETR(II)
ETR(I)/ETR(II)
B
0%
PPFD (μmol m
2
s
Time (min)
1)
Fig. 8 Light intensity dependence of the ETR(I)/ETR(II) ratio in 20, 2.7
and 0% O2 in WT (filled symbols) and pgr5 (open symbols) leaves.
Measurements were made for the PPFDs ranging from 0 to 280 mmol
photons m2 s1 and at a CO2 concentration of 390 p.p.m. The values
represent the mean ± SD (n = 3).
Fig. 9 Changes in the ETR(I)/ETR(II) ratio in WT (A) and pgr5 (B)
leaves in fluctuating light. The same light treatment protocol as in
Fig. 1 was used. Measurements were made at 20% O2 and 390 p.p.m.
CO2. The values represent the mean ± SD (n = 5–6).
pgr5
WT
Effects of O2 concentration on responses of
electron transport to fluctuating light
Responses of Y(II) to the fluctuating light were measured at
2.7% and 0% O2 (Fig. 10). In pgr5, Y(II) at the end of the LL
period decreased only slightly with the cycle at 2.7% O2. The
decrease in Y(II) in LL was smaller still at 0% O2. Moreover, in
contrast to the gradual decrease in Y(II) in the HL period at 20%
O2, Y(II) in HL increased with the cycle at 2.7% and 0% O2.
Similarly, in the WT, Y(II) at the end of the LL period did not
decrease with the cycle at 2.7% or 0% O2, whereas Y(II) at the
end of the LL period decreased at 20% O2. Y(II) in the HL period
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2.7% O2
Y(II)
2.7% O2
0% O2
0% O2
Y(II)
Fig. 1 was used. Consistent with the data in Fig. 6, the ETR(I)/
ETR(II) ratio in the pgr5 plants was lower in the HL period than in
the LL period, whereas WT plants showed a higher ETR(I)/ETR(II)
ratio in the HL period than in the LL period. In the WT, the
ETR(I)/ETR(II) ratio reached a maximum at 15 s after each transfer from the LL period to the HL period, and, within 2 min,
decreased slowly toward the steady-state value. The maximal
value at 15 s after the transfer gradually increased with the
cycle, while the steady-state value slightly decreased. In contrast,
in pgr5, the ETR(I)/ETR(II) ratio rapidly decreased to the minimum at 15 s after the transfer from the LL period to the HL
period. The minimal value gradually increased with the cycles.
Also the ratio at the last data point in the HL period increased
with the cycle. The peak value in the 2 min LL period gradually
increased. In each of the LL periods, the ratio decreased.
Time (min)
Time (min)
Fig. 10 Effects of low O2 concentrations (2.7%, diamond; 0%, triangle)
on responses of Y(II) to the fluctuating light in the WT (fillsed symbol)
and pgr5 (open symbol). The fluctuating light treatment was the same
as that used for Fig. 1. Measurements were made at 390 p.p.m. CO2.
The values represent the mean (n = 4–6).
at 2.7% and 0% O2 continued to increase with the cycle and did
not reach the steady-state values within 42 min.
We assessed the degrees of photoinhibition after the treatment with fluctuating light for 42 min at 0, 2.7 or 20% O2 and
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
Responses of alternative electron flows to fluctuating light
ΔAmax
B
Y(NA)30
C
Fv/Fm
1-qL30
D
Relative value
A
F
G
H
42m
in
e
42m
in
bef
ore
bef
or
42m
in
bef
ore
42m
in
bef
ore
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
bef
ore
42m
in
Relative value
E
Fig. 11 Effects of low O2 concentrations (2.7%, a–d; 0%, e–h) on changes in photosynthetic parameters after the fluctuating light treatment in
the WT (black bar) and pgr5 (white bar). Measurements were made in the same manner as in Fig. 4 and at 390 p.p.m. CO2. Error bars represent
the SD (n = 4–8).
dark treatment for 30 min (Fig. 11) using the same experimental protocol that was used for the data shown in Fig. 4. The pgr5
plants showed little photoinhibition of PSI at 2.7% and 0% O2
compared with that at 20% O2 (cf. Fig. 4e and Fig. 11a and e).
The marked increases observed in Y(NA)30 and 1 – qL30 after
the light treatment at 20% O2 were greatly suppressed at low
O2, although the increases were consistently observed not only
in pgr5 but also in the WT.
Discussion
Fluctuating light as a stress factor causing
photodamage
How plants use sunflecks—pulses of light at high intensity—is a
topic that has attracted the attention of researchers for decades
(Allee 1926, Evans 1956). There have been many laboratorybased mechanistic studies as well as field-oriented studies
focusing on photosynthetic responses to fluctuating light. For
instance, photosynthetic responses of plants grown in controlled fluctuating light and those grown in constant light
were compared (Yin and Johnson 2000, Alter et al. 2012,
Suorsa et al. 2012). The responses were also compared between
plants grown in natural fluctuating light in a forest understorey
and those grown in an open field site (Knapp and Smith 1989).
In most of these studies, photosynthetic responses to a single
light pulse were examined. However, in natural environments,
such as the forest understorey, light fluctuates more frequently,
as many researchers quantified (Pearcy 1983, Chazdon 1988,
Vierling and Wessman 2000). Although there have been some
pioneering studies (Alter et al. 2012, Suorsa et al. 2012), our
knowledge of the photosynthetic responses to fluctuating light
is still poor.
It is important to choose appropriate fluctuating light regimes
for studying plant responses to fluctuating light. The light environment in the forest understorey changes drastically due to
sunflecks. Most sunsflecks are less than several minutes in
length, and have a PPFD more than several-fold that of LL periods
(Pearcy 1983, Koizumi and Oshima 1993, Vierling and Wessman
2000). Recently, Suorsa et al. (2012) grew several mutant lines of
A. thaliana in the light alternately changing from LL at 50 mmol
photons m2 s1 for 5 min to high HL at 500 mmol photons
m2 s1 for 1 min, and successfully elucidated a role for the
PGR5 protein in acclimation to the fluctuating light. In the present study, we used a fluctuating light regime with the same
durations of HL and LL. The duration we adopted was 2 min
because the photosynthetic parameters change most drastically
upon the change from the LL to the HL period in the first 2 min in
A. thaliana plants. Thus, the light fluctuation in the 2 min intervals would subject the plants to the most stressful situation.
Next, we chose the intensity of the HL. The HL at 240 mmol
photons m2 s1 was strong enough, but did not induce photoinhibition when given continuously. The present results indicate
that the fluctuating light we adopted was suitable for analysis of
the effects of fluctuating light on photoinhibition. The results
clearly showed that light fluctuation itself is a very effective stress
factor causing photodamage. We propose the term ‘fluctuating
light photoinhibition’ and the target is mainly PSI, as has been
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
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already indicated by pioneering studies (Munekage et al. 2002,
Munekage et al. 2008, Suorsa et al. 2012).
Effects of short-term fluctuating light on the
photosynthetic electron transport system
The decreases in the photochemical quantum yield of PSII, Y(II),
of the WT and pgr5 plants occurred soon after the start of
the fluctuating light treatment. Y(II) of pgr5 decreased more
drastically (Fig. 1). Moreover, these plants showed photoinhibition of PSI by the fluctuating light treatment. In particular, the
extent of PSI photoinhibition in pgr5 was marked. It should be
noted that this was not the result of the long-term effect of the
fluctuating ‘growth’ light (Suorsa et al. 2012) but that of treatment for a short period. It has been reported that PSI of pgr5
was sensitive to light (Munekage et al. 2002, Munekage et al.
2008). In the present study, we found that PSI activity in pgr5
was limited by the acceptor-side reactions: Y(NA) was higher
than Y(ND) over the entire PPFD range examined (Fig. 3).
What component/event in the PSI acceptor side limited the
photochemical reaction and thereby caused PSI photoinhibition?
The crucial difference between the fluctuating light and constant
light was that fluctuating light included a LL period, during which
photosynthetic activities were lower than those during the HL
period. When leaves were in the LL, various reactions that had
occurred in response to HL, including the de-epoxidation of
violaxanthin and protonation of the PsbS protein, would be
relaxed to some extent. In the HL period, the thylakoid lumen
acidification would not be enough for down-regulation of LEF via
the photosynthetic control of plastoquinol re-oxidation at the
Cyt b6/f complex (Rott et al. 2011, Suorsa et al. 2012), particularly
in pgr5. Therefore, especially in pgr5, when every HL period
started, the thylakoids in the more or less relaxed state would
cause a gush of electron flow to PSI, leading to prompt reduction
of electron acceptors, O2 photoreduction and formation of ROS.
From preceding studies (Sonoike 1996, Sonoike et al. 1997, Choi
et al. 2002), it is clear that the photoinhibition of PSI involves ROS
and thereby requires O2.
It is noteworthy that the PSI photoihibition occurred even in
the WT plants due to the fluctuating light; the photodamage by
fluctuating light is not a phenomenon specific only to pgr5.
The response to fluctuating light effectively
avoiding photoinhibition
The large Y(NA) means a high level of PSI acceptor-side limitation. This is not necessarily directly associated with photoinhibition of PSI. As clearly shown in Fig. 4, pgr5 treated in
continuous HL showed little photoinhibition of PSI, although
Y(NA)30 and 1 – qL30, measured at the end of the illumination
of 30 mmol photons m2 s1 for 2 min, just after the constant
HL for 42 min, were very high. On the other hand, when fluctuating light was applied for 42 min, PSI photoinhibition
occurred in both the WT and pgr5, in addition to the increases
in Y(NA)30 and 1 – qL30. The extent of the damage was markedly greater in pgr5. The large difference in the PSI
998
photoinhibition between the WT and pgr5 plants indicates
that the WT had mechanisms to cope with rapid light fluctuations. We hypothesized that this would be related to photosynthetic alternative electron flows interacting with PSI because
PSI was first photoinhibited in the fluctuating light. Previous
studies reported that, in pgr5, the CEF-PSI via the putative FQR
was impaired (Munekage et al. 2002, Munekage et al. 2004,
DalCorso et al. 2008). However, some other studies reported
that pgr5 possessed CEF-PSI capacity (Nandha et al. 2007, Joliot
and Johnson 2011). We tried to assess the activities of the alternative electron flows in pgr5.
Several methods have been used to quantify the rate of the
CEF-PSI (for a review, see Kramer et al. 2004a). In LEF, the rates
of electron transfer through PSII should equal that through PSI
(Klughammer and Schreiber 1994) or the Cyt b6/f complex
(Klughammer and Schreiber 1994, Sacksteder and Kramer
2000). Thus, the relationship between some factors associated
with the electron flow and the LEF should be changed when the
activity of the CEF-PSI becomes substantial. In turn, from the
increased ratios of these factors to LEF, the rate of the CEF-PSI
would be assessed. The possible factors would include the
proton to electron stoichiometry (Sacksteder et al. 2000), electrochromic shift of carotenoid pigments due to the formation
of an electric field across the thylakoid membrane (Joliot and
Joliot 2002, Joliot and Joliot 2005, Joliot et al. 2004, Sacksteder
and Kramer 2000) and the proportion of overall photosynthetic
energy storage assessed by the photoacoustic method (Herbert
et al. 1990, Joet et al. 2002). More directly, measurements of
post-illumination re-reduction kinetics of P700+ after red + farred actinic light (Fan et al. 2007), or after a far-red illumination
(Maxwell and Biggins 1976, Joet et al. 2002, Chow and Hope
2004), have been conducted. The transient rise in the fluorescence level after turning off the actinic light has also been
measured as a parameter reflecting the activity of CEF-PSI
(Asada et al. 1993, Burrows et al. 1998). However, it is not feasible to measure the absolute rate of the CEF-PSI in situ with
these techniques. Instead, we used the ETR(I)/ETR(II) ratio as an
indicator of the CEF-PSI activity (Fig. 6). Because the linear
electron transport rate through PSII can be quantified (Genty
et al. 1989), if the ratio is properly obtained, the rate through PSI
may be quantified. For this purpose, we measured leaf absorptance and the share of absorbed light energy allocated to PSII
(fPSII) (Table 1). Very recently, Kou et al. (2013) estimated the
activity of PSI-CEF at saturating CO2 based on measurements of
the O2 evolution rate and PSI quantum yields. However, they
did not measure fPSII.
Solving the equation, Y(O2) = IAfPSIIY(II)/4, where IA is the
absorbed PPFD (Genty et al. 1989), we obtained the share of
absorbed light energy allocated to PSII. In spite of the fact that
the plants were grown under constant illumination, the share in
pgr5 was 35%, while the WT showed almost equal sharing of light
energy between PSI and PSII (Fig. 5B). Furthermore, the contribution of the state transition to the energy share was small over
the range from low to high PPFD in both plants (Fig. 7), in
agreement with the studies reporting that the state transitions
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Responses of alternative electron flows to fluctuating light
in higher plants were not marked (Pesaresi et al. 2011). These
results indicate that the share of light energy allocation to PSI was
much greater than that to PSII in pgr5. In the light–response
curve shown in Fig. 3, Y(I) in pgr5 started to decrease from
very low PPFDs, whereas that in the WT was relatively high up
to a PPFD of approximately 250 mmol photons m2 s1 and
started to decrease with a further increase in PPFD. In pgr5, a
limitation of electron flow through PSI due to this decrease in
Y(I) at low PPFD would be compensated by the increase in the
share of absorbed light energy allocated to PSI. This was also
supported by the fact that the Chl a/b ratio was greater in the
pgr5 leaves than that in the WT by 0.4. Previous studies reported
that growth of pgr5 was similar to that of the WT in both low
light (Munekage et al. 2008) and moderate light (Suorsa et al.
2012). However, in fact, energy sharing between the two photosystems and the composition of Chl proteins would be markedly
changed in pgr5. The calculation of the ETR using properly measured fPSII may be a useful method to estimate CEF-PSI.
When white light was used for the actinic light, maximum
Y(O2) values for non-stressed leaves of C3 plants approached
0.105 (Björkman and Demmig 1987). In this study, Y(O2)
decreased with the absorbed PPFD at low PPFDs because red
light was used for the actinic light. However, when the maximum Y(O2) was obtained by extrapolating the line in Fig. 5B to
a Y(II) of 0.81, the value for the WT was 0.09, a value within the
range of the data for C3 species (Björkman and Demmig 1987).
From the light energy allocation to PSI and the changes in
the ETR(I)/ETR(II) ratio measured in the constant and fluctuating light, we suggest that pgr5 plants possessed CEF-PSI activities because the ratios at low PPFDs and LL in the fluctuating
light were far greater than 1 (Figs. 6, 9). These results also
indicate that, under the growth light conditions at a PPFD of
100 mmol photons m2 s1, pgr5 drove CEF-PSI continuously
(Nandha et al. 2007). On the other hand, the ETR(I)/ETR(II)
ratio in the WT increased with the increase in PPFD. This suggests that the CEF-PSI functions not only during photosynthetic
induction (Makino et al. 2002, Joliot and Joliot 2002, Joliot and
Joliot 2005, Joliot and Joliot 2006, Fan et al. 2007) but also under
steady-state conditions in high light.
There are some O2-dependent pathways besides the WWC,
and they may contribute to the photodamage by the fluctuating light. Photorespiration is one of the O2-dependent pathways. When CO2 and O2 concentrations were 800 p.p.m. and
20%, respectively, in the leaf chamber where the effect of photorespiration was suppressed to a considerable extent, Y(II) of the
WT and pgr5 showed responses similar to those at 390 p.p.m.
and 20% (Supplementary Fig. S1). Plastid terminal oxidase
(PTOX) is also proposed to be associated with O2 consumption,
in the reaction called chlororespiration. The PTOX is a plastoquinol oxidase, and is able to transfer electrons from PQ to O2.
Thus, chlororespiration can be a source of ROS generation.
However, PTOX is suggested to play an important role in
chloroplast biogenesis rather than in stress responses (Rosso
et al. 2006). Furthermore, in plants grown under normal conditions, PTOX is present at about only 1% of the level of the D1
protein that houses the PSII reaction center (Lennon et al.
2003). Therefore, the contributions of photorespiration and
chlororespiration to the photodamage caused by the fluctuating light would be small, if any.
In both the constant and fluctuating light, pgr5 appeared to
show WWC activities (Fig. 8). When the light dependence of
the ETR(I)/ETR(II) ratio was measured at 2.7% or 0% O2, the
ratios in pgr5 at a PPFD >100 mmol photons m2 s1 were
greater than those measured at 20% O2. This suggests that at
least some fraction of electrons that flowed through the WWC
at 20% O2 would flow through the CEF-PSI at low O2 concentrations, resulting in increases in the ETR(I)/ETR(II) ratio. The
ETR(I)/ETR(II) ratios in the WT and pgr5 were highest at 2.7%
O2 rather than at 0% O2 for all the PPFD levels examined
(Fig. 8). The reasons for this are unknown.
For WT plants in the fluctuating light, the ETR(I)/ETR(II)
ratio in the HL periods rapidly increased immediately after
each transition from the LL to the HL period, attained maximal
levels, then decreased and attained steady-state values within
the HL periods. Although the steady-state values decreased in a
stepwise fashion with the cycle, the maximal levels were almost
constant. In contrast, in pgr5, the ratio rapidly decreased immediately after each transition from an LL to a HL period and
then attained the minimum levels within the HL periods. Under
these conditions, PSI of pgr5 would be more sensitive to the
damage due to the fact that PSI capacity was not able to
manage the gush of electron flow caused by the rapid increase
in PPFD. However, at low O2 concentrations, Y(II) in the LL
period in fluctuating light did not decrease with the cycles
(Fig. 10), and no photoinhibition of PSI occurred after the
light treatment (Fig. 11). These results indicate that an increase
in the activity of the CEF-PSI at low O2 concentrations led to
relaxation of the acceptor-side limitation of PSI, resulting in
acceleration of the linear and/or the other electron flows.
Therefore, we conclude that the CEF-PSI is essential to cope
efficiently with the rapid increase in PPFD and prevent photoinhibition of PSI caused by the fluctuating light. Furthermore,
our data indicate that the CEF-PSI could be regulated by O2.
The enhancement of the CEF-PSI by low O2 is probably attributable to suppression of the electron flow to O2 at low O2. As
the activity of the CEF-PSI cannot be properly regulated in pgr5,
a considerable number of electrons inevitably flow to O2,
leading to ROS formation and thereby PSI photoinhibition.
From these findings, it is suggested that, in the WT, electron
flow to O2 can be controlled by regulating engagement of
alternative electron flows including CEF-PSI in such a way
that the photooxidative damage is minimized even at 20% O2.
Apparently, the difference in the responses to the HL periods
between the WT and pgr5 disappeared with the cycles (Fig. 9).
However, this was due to underestimation of Y(I) that was
calculated from the maximal P700 signal, denoted as Pm, because Pm was measured before the fluctuating light treatment.
Since Pm decreased greatly in pgr5 and slightly in the WT due to
PSI photoinhibition, there were large differences in the ETR(I)/
ETR(II) ratio in the HL period between the WT and pgr5 when
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Pm values corrected for the photodamaged PSI were used (see
Supplementary Fig. S2).
In A. thaliana, NDH-CEF has been suggested to play a complementary role, since the NDH-CEF is not essential for photosynthesis at least under ordinary laboratory conditions, and
NDH-deficient mutants of A. thaliana grow similarly to the
WT (Munekage et al. 2002, Munekage et al. 2004, Okegawa
et al. 2008). We measured the responses of an NDH-deficient
mutant, crr2-2, to the fluctuating light under the same conditions as those use for Fig. 1 (Supplementary Fig. S3). The
results in crr2-2 were almost identical to those in the WT;
Y(II) decreased with the cycles, showing similar changes in
the ETR(I)/ETR(II) ratio, and PSI was slightly photoinhibited
by the fluctuating light treatment. The light dependence of
the ETR(I)/ETR(II) ratio exhibited trends similar to those in
the WT. Moreover, at any PPFD, the ratios at 2.7% and 0% O2
were higher than that at 0% O2 (Supplementary Fig. S4). Thus,
we conclude that the NDH-CEF would not contribute to the
response to the fluctuating light. It is noteworthy, however, that
ETR(I)/ETR(II) ratios in the WT and pgr5 at the two lowest
PPFD levels were somewhat greater than those at 35 mmol
m2 s1 (Fig. 8). This was not the case in crr2-2, although we
did not measure the ratios at very low PPFDs for crr2-2. These
differences may indicate that NDH-CEF in the WT and pgr5
operated at very low PPFDs, as suggested for Oryza sativa
(Yamori et al. 2011) and Marchantia polymorpa (Ueda et al.
2012). It is necessary to conduct detailed measurements including fPSII with crr2-2.
Concluding remarks and future scopes
PSI of the pgr5 plant was sensitive, as previously reported
(Munekage et al. 2008), due to the large acceptor-side limitation
of PSI. pgr5 was particularly sensitive to the fluctuating light,
and showed marked photoinhibition of PSI. In this study, we
clearly elucidated that pgr5 can drive CEF-PSI in low light.
Namely, pgr5 not only possesses the capacity for CEF-PSI
(Nandha et al. 2007) but actually drives the CEF-PSI at low
PPFDs. However, its capacity dramatically decreases with the
increase in the PPFD, supporting the view that the PGR5 protein is involved in the redox control of PSI (Nandha et al. 2007).
The general message of this study is that the CEF-PSI is essential for effective responses to drastic light fluctuation. When
plants are exposed to drastic fluctuation in PPFD in the field,
they would activate the CEF-PSI more than the WWC to accommodate the electron flows and thereby avoid the risk of
photo-oxidative damage. We also found that the fluctuation in
PPFD is a potent stress factor, even when the PPFD level in the
HL periods is moderate.
Plants grown in the forest understorey are exposed to drastic
fluctuations in PPFD. If they are able to acclimate to such
fluctuating light conditions, one of the mechanisms would be
an enhancement of the ability for appropriate regulation of the
activity of the CEF-PSI in response to light fluctuation. This
would be achieved by the increase in the proportion of the
1000
PSI complex with the PGR5 protein. Plants may be able to
avoid photodamage to PSI by altering the ratio of the two
photosystems in the thylakoid membranes (Jahns and Junge
1992, Yin and Johnson 2000, Suorsa et al. 2012) as observed
in pgr5 grown in constant light in this study. We are currently
examining whether the photosynthetic apparatus in the WT
acclimates to the drastically fluctuating growth light to actually
become resistant to the fluctuating light.
Materials and Methods
Plant materials
Arabidopsis thaliana WT (ecotype Columbia gl1) and pgr5
mutant (Munekage et al. 2002) plants were pot grown in a
growth cabinet with white fluorescent light at 90–100 mmol
photons m2 s1 for an 8 h photoperiod at room temperature
of 23 C and relative humidity of 60%. Plants were irrigated 2–3
times weekly and were fertilized with Hyponex 6-10-5 solution
(Hyponex Japan) diluted to 1 : 1,000 strength at every irrigation
from 2 weeks after germination. Mature rosette leaves from 7to 9-week-old plants were used in the experiments.
Chl fluorescence and 830 nm absorbance change
measurements
Chl fluorescence and absorption changes at 830 nm were measured simultaneously using a Dual-PAM-100 (Chl fluorescence
and P700 absorption analyzer equipped with a P700 dual wavelength emitter at 830 and 875 nm; Walz) with the intact leaf in a
hand-made leaf chamber. The CO2 concentration in the leaf
chamber was monitored with a LI-6400 (Li-Cor). The O2 concentration in the air was controlled by mixing N2 gas and O2 gas
using mass flow controllers. Saturation pulses (SPs) from red
light-emitting diodes (LEDs; >8,000 mmol photons m2 s1,
400 ms duration) were applied to determine the maximum
Chl fluorescence with closed PSII centers in the dark (Fm) and
in the actinic light (Fm0 ). The maximum photochemical quantum yield of PSII (Fv/Fm) and effective quantum yield of PSII
[Y(II)] were calculated as (Fm – F0)/Fm and (Fm0 – Fs0 )/Fm0
(Genty et al. 1989), respectively, where Fs0 is the steady-state
Chl fluorescence level in the actinic light from red LEDs with a
wavelength peak at 635 nm, in which chloroplast avoidance
movement does not occur and has no effect on assessment of
non-photochemical quenching components (Cazzaniga et al.
2013). The coefficient of non-photochemical quenching, qN,
was calculated as (Fm – Fm0 )/(Fm – F00 ). F00 is the minimal fluorescence yield in actinic light and was estimated using the approximation of Oxborough and Baker (1997) as F0/(Fv/Fm + F0/
Fm0 ). Two other PSII quantum yields, Y(NPQ) and Y(NO) (Genty
et al. 1996, Kramer et al. 2004b), which represent the regulated
and non-regulated energy dissipation at PSII centers, respectively,
and add up to unity with the photochemical quantum yield [i.e.
Y(II) + Y(NPQ) + Y(NO) = 1], were also used. Y(NPQ) and
Y(NO) were calculated as Fs0 /Fm0 – Fs0 /Fm and Fs0 /Fm, respectively
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
Responses of alternative electron flows to fluctuating light
(Hendrickson et al. 2004, Klughammer and Schreiber 2008a). The
coefficient of photochemical quenching, qL, a measure of the
fraction of open PSII reaction centers, based on the ‘lake model’
of PSII antenna pigment organization, was calculated as (Fm0 –
Fs0 )/(Fm0 – F00 )F00 /Fs0 (Kramer et al. 2004b).
In the Dual-PAM-100, P700+ was monitored as the absorption difference between 830 and 875 nm in transmission mode.
In analogy to Chl fluorescence yield, the quantum yield of PSI
was determined using the SP method (Klughammer and
Schreiber 1994, Klughammer and Schreiber 2008b). The maximum level of P700 signal (P700 fully oxidized) in the dark,
called the Pm, was determined by application of an SP in the
presence of far-red light at the wavelength of 720 nm. The zero
P700 signal, P0, was determined when complete reduction of
P700 was induced after the SP in the absence of far-red light. Pm0
is the maximal P700 signal in the presence of actinic light
induced by the SP. The photochemical quantum yield of PSI,
Y(I), was calculated from the complementary PSI quantum
yields of non-photochemical energy dissipation, Y(ND) and
Y(NA), respectively: Y(I) = 1 – Y(ND) – Y(NA). Y(ND) corresponds to the fraction of P700 that is already oxidized by actinic
light, and Y(NA) corresponds to the fraction of P700 that
cannot be oxidized by an SP to the overall P700. These calculations were made according to Klughammer and Schreiber
(2008b). To oxidize the intersystem electron carriers, far-red
light was applied from 100 ms before the start of the SP to its
cessation. As shown in Fig. 2, Y(I) did not decrease under constant HL. Our preliminary checks showed that an SP of 400 ms
duration was enough to induce maximal P700+ oxidation level
and to obtain complete reduction of the level of P700 after the
SP. Photodamage by the SP did not occur.
The proportions of the non-photochemical quenching components were determined from the relaxation kinetics of the
variable fluorescence (Fv) in the absence of actinic light for
30 min (Quick and Stitt 1989, Walters and Horton 1991).
Relaxation of Fv was monitored with SPs given every 100 s to
the leaf. The intervals of 100 s were sufficient to eliminate an
effect of the SP on Fv relaxation. The fast-relaxing component of
fluorescence quenching was assigned to the energy-dependent
mechanism (qE), the intermediate relaxing component was assigned to the state transition (qT) and the slow relaxing component was assigned to the photoinhibitory processes (qI). For
quantification of qE, qT and qI, the semi-logarithmic plot of Fv
vs. time was analyzed considering the relationship (1 – qN) = (1
– qE)(1 – qT)(1 – qI).
We estimated the electron transport rate through PSI
[ETR(I)] and PSII [ETR(II)] simultaneously. In this study, a
source of artifacts should be considered for a comparison of
ETR(I) with ETR(II). With the blue measuring light, the Chl
fluorescence signal mainly emitted from the upper layer of
the mesophyll cells closest to the emitter detector unit was
detected, while the P700 signal detected was more generally
from the whole leaf tissue. In HL, these upper cells would be
more prone to light saturation of photosynthesis and photoinhibition than the cells in the deeper layer (Terashima et al.
2009, Oguchi et al. 2011a, Oguchi et al. 2011b). To prevent this
preferential light saturation of photosynthesis and photoinhibition near the leaf surface effectively, red light at 635 nm peak
wavelength instead of blue light was used as the actinic light.
The red actinic light at this wavelength reaches the deeper cell
layers, and would cause more even light saturation of photosynthesis and photoinhibition than the blue actinic light.
PSI fluorescence may contribute to total leaf fluorescence
(Pfundel 1998, Rappaport et al. 2007). In this study, blue light at
460 nm peak wavelength was used as the measuring light,
except for the data shown in Fig 5; see below. The blue measuring light excites PSII more than PSI. In addition, according to
Pfundel et al. (2013), emission of PSI fluorescence by the A.
thaliana leaves is low irrespective of growth PPFD levels.
The P700 signal can be interfered with by the absorbance
changes of plastocyanin. Up to 10% of the P700 difference in
absorption signal measured by the Dual-PAM instrument may
be attributable to plastocyanin, which shows considerable absorption at both 830 and 870 nm (Kirchhoff et al. 2004). Livingston
et al. (2010) compared the results with the Dual-PAM system and
those using the two-wavelength deconvolution method (A of
820–950 nm) described by Oja et al. (2004) and concluded that
absorbance changes from plastocyanin or other components
may not substantially affect the P700 measurements.
Measurements of the share of absorbed light
energy allocated to PSII
To estimate the share of absorbed light energy allocated to PSI
and PSII, simultaneous measurements of O2 evolution and Chl
fluorescence in the leaf were made at 23 C using a leaf disk
oxygen electrode system (LD2, Hansatech) and a Chl fluorometer (PAM-2500, Walz).
Leaf segments were placed in the chamber of the leaf disk O2
electrode. When the steady-state rate of O2 evolution was attained, the quantum yield of PSII photochemistry [Y(II)] was
measured. The irradiance of the actinic light was increased in a
stepwise manner. A Björkman-type lamp equipped with a red
color filter of the wavelength centered at 635 nm was used as
the light source. The red light was used to mimic the spectrum
of the actinic light of the Dual-PAM system. PPFD was altered
with neutral density filters (Toshiba). The air in the chamber
contained about 5% CO2 and 15% O2. The quantum yield of O2
evolution [Y(O2)] of the leaf was calculated by dividing the rate
of gross O2 evolution per leaf area (mmol O2 m2 s1) by absorbed PPFD. The absorptance of the leaf was measured with a
hand-made integrating sphere, whose inside was coated with
BaSO4, and a quantum sensor (LI-190SA, Li-Cor). When fPSII, the
share of absorbed light energy allocated to PSII, is <0.5, the
relationship between the quantum yield of O2 evolution at
saturating CO2 [Y(O2)] and that of PSII electron transport
[Y(II)] can be expressed as Y(O2) = IAfPSIIY(II)/4, where IA
is the absorbed PPFD (Genty et al. 1989).
This equation, which compares gross O2 evolution from the
whole tissue with Y(II) obtained from the shallow part of the
Plant Cell Physiol. 55(5): 990–1004 (2014) doi:10.1093/pcp/pcu033 ! The Author 2014.
1001
M. Kono et al.
mesophyll, may lead to uncertainty in fPSII. The error in fPSII gives
rise to uncertainty in ETR(II). Since fluorometrically estimated
ETR(II) tends to be underestimated compared with that calculated from the gross O2 evolution rate, especially at high PPFDs,
the ETR(I)/ETR(II) ratio calculated with fluorometrical ETR(II)
would be overestimated (Kou et al. 2013). To obtain a Chl
fluorescence signal from the deeper mesophyll cells with the
PAM-2500, we used red light with a peak at 630 nm as the
measuring light. The use of the red measuring light, rather
than blue light, would minimalize the error in estimation of
the ETR(II), particularly that at relatively low PPFDs. Thus, we
used data points obtained at low PPFDs. Effects of fluorescence
from PSI would be small at low PPFDs.
Determination of Chl content
Chl a and b contents were determined according to Porra et al.
(1989).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Ministry of Education, Culture,
Sports, Science and Technology, Japan [Grant-in-Aid for
Scientific Research on Innovative Areas (21114007)].
Acknowledgments
We thank Professor Toshiharu Shikanai for providing the pgr5
and wild-type Columbia type gl1 seeds, and Professors Kintake
Sonoike, Masahiko Ikeuchi, Chikahiro Miyake and Toshiharu
Shikanai, and anonymous reviewers for helpful discussions.
We would like to dedicate this paper to the late Professor
Kozi Asada who passed away on 15 December 2013.
Disclosures
The authors have no conflicts of interest to declare.
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